Addition reaction products of conjugated sunflower-oil fatty acids and tall-oil fatty acids with maleic anhydride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2 mM L-glutamine, 1 (v/v) % Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and 10 (v/v) % heat-inactivated fetal bovine serum (DMEM-10, culture medium). During the treatments, the serum content of the medium was reduced to 5 (v/v) % (DMEM-5).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (induction of rat liver enzymes by Phenobarbital and β-naphthoflavone)

Test concentrations with justification for top dose:

Assay 1, 3h treatment with metabolic activation:
5000, 3750, 2500, 1250, 625, 312.5, 156.25, 78.13 and 39.06 μg/mL
Assay 1, 3 h treatment without metabolic activation:
2000, 1000, 750, 500, 375, 250, 125, 62.5 and 31.25 μg/mL
Assay 2, 3h with treatment with metabolic activation:
2500, 2187.5, 1875, 1562.5, 1250, 625, 312.5, 156.25, 78.13 and 39.06 μg/mL
Assay 2: 20h treatment without metabolic activation:
1000, 500, 375, 312.5, 250, 187.5, 125, 62.5 and 31.25 μg/mL
Assay 3, 3h with treatment with metabolic activation:
2000, 1500, 1000, 875, 750, 625, 500, 375, 250, 125, 62.5 and 31.25 μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: appropriate for formulation and its dilution; compatible to the test system

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

ethylmethanesulphonate

Remarks:

Ethylmethanesulfonate (EMS) dissolved in DMEM, final concentration of 0.4 μL/mL (28-hour harvesting time) or 1.0 μL/mL (20-hour harvesting time)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

Cyclophosphamide (CP) dissolved in sterile physiological saline solution (0.9% NaCl infusion), final concentration of 6.0 μg/mL.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 and 20 hours
- Fixation time (start of exposure up to harvest of cells): 20 and 28 hours

SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.2 μg/mL)
STAIN (for cytogenetic assays): 5 % Giemsa solution

NUMBER OF CELLS EVALUATED: 200 cells (metaphases)/dose

DETERMINATION OF CYTOTOXICITY
- Method: determination of cell concentration by use of a haemocytometer

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolarity of the treated cultures.

Statistics:

For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see Table 1 and 2 ("Additional information on results")

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no changes
- Effects of osmolality: changes observed ≥ 3750 μg/mL in the experiment with metabolic activation (only in Assay 1).
- Insolubility: ≥ 312,5 and ≥ 375 μg/mL with and without metabolic acitvation, respectively. Insolubility was detected at the end of the treatment period in the final treatment medium.

Any other information on results incl. tables

Table 1: Summarized results of the concentration selection cytotoxicity assays A and B

	Dose(μg/mL)	Testgroup without S9-mix Relative survival (%) (Assay A*/B**)	Testgroup with S9-mix Relative survival (%) (Assay A*/B**)
Untreated control		92/119	112/119
Vehicle control 1%(v/v) DMSO		100/100	100/100
Vehicle control 2% v/v DMSO		100/100	100/100
WS400104	5000	1/0	18/3
	2500	23/0	62/2
	1250	34/0	52/51
	625	12/0	39/44
	312,5	52/7	47/72
	156,25	71/75	69/96
	78,13	88/97	81/93
	39,06	83/91	95/102

*Time of Treatment/Sampling: 3h/20h with and without activation

**Time of Treatment/Sampling: 20h/28h without activation, 3h/28h with activation

Table 2: Summary table of Chromosome Aberration Assay 1

Concentration (μg/mL)	Time of Treatment / Sampling	Relative Survival # (%)	Mean aberrant cells### %
WS400104 without metabolic activation (-S9)
Vehicle(solvent)control	3h/20h	100	3.0
2000	3h/20h	19	NE
1000	3h/20h	25	NE
750	3h/20h	10	NE
500	3h/20h	32	5,0
375	3h/20h	58	NE
250	3h/20h	64	4,5
125	3h /20h	93	3,5
62.5	3h/20h	92	NE
31.25	3h/20h	110	NE
Positive control	3h/20h	84	11,0**

WS400104 with metabolic activation (+S9)
Vehicle(solvent)control	3h/20h	100	3.0
5000	3h/20h	0	NE
3750	3h/20h	27	2,7
2500	3h/20h	0	NE
1250	3h/20h	0	NE
625	3h/20h	40	12,4**
312,5	3h/20h	87	3,5
156,25	3h/20h	87	5,0
78,13	3h/20h	94
39,06	3h/20h	88
Positive control	3h/20h	66	96.8***

Vehicle (solvent) control: 1% (v/v) DMSO without (Assay –S9), 2% (v/v) DMSO (Assay +S9)

Positive control (-S9): Ethyl methanesulfonate, 1μL/mL; Positive control (+S9): Cyclophosphamide, 6μg/mL

NE: not evaluated

#: compared to the vehicle (solvent control)

##: in the final treatment medium at the end of the treatment

###: excluding gaps

**: p<0.01 comparing numbers of aberrant cells excluding gaps with corresponding negative control

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

Table 3: Summary table of Chromosome Aberration Assay 2

Concentration(μg/mL)	TimeofTreatment/Sampling	Relative # Survival (%)	Mean%aberrantcells###
WS400104withoutmetabolicactivation(-S9)
Vehicle(solvent)control	20h/28h	100	3,0
1000	20h/28h	0	NE
500	20h/28h	0	NE
375	20h/28h	0	NE
312,5	20h/28h	0	NE
250	20h/28h	15	NE
187,5	20h/28h	13	2.0
125	20h/28h	57	1.0
62,5	20h / 28h	91	2,0
31.25	20h/28h	91	NE
Positivecontrol	20h/28h	63	21,4***
WS400104withmetabolicactivation(+S9)
Vehicle(solvent)control	3h/28h	100	3.0
2500	3h/28h	59	NE
2187,5	3h/28h	53	NE
1875	3h/28h	37	NE
1562,5	3h/28h	46	NE
1250	3h/28h	38	1,5
625	3h/28h	47	6,0
312,5	3h/28h	49	2,5
156,52	3h/28h	87	2,0
78,13	3h/28h	94	3,0
39,06	3h/28h	75	NE
Positivecontrol	3h/28h	58	12,6***

Vehicle (solvent) control: 1% (v/v) DMSO

Positive control (-S9): Ethyl methanesulfonate, 0.4μL/mL; positive control (+S9): Cyclophosphamide, 6 μg/mL

NE: not evaluated

#: compared to the negative (solvent control)

##: in the final treatment medium at the end of the treatment

###: excluding gaps

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

In Assays 1 and 2, none of the treatment doses caused a significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation except for 625 μg/mL concentration in Assay 1 with metabolic activation, in one replicate. This was not repeatable in Assay 2 (although elevated aberration frequency was observed at the same concentration), nor did the higher concentration assessed in Assay 1 with metabolic activation give a positive response. There was no evidence of any dose response in either assay.

Because of the equivocal results observed in Assay 1 with metabolic activation, an additional experiment with metabolic activation (Assay 3) was performed - with a more closely spaced concentration range, but otherwise using the same treatment and harvesting time as in Assay 1.

Table 4: Summary table of Chromosome Aberration Assay 3 with metabolic activation

Concentration (μg/mL)	Time of Treatment / Sampling	Relative Survival#(%)	Mean % aberrant cells###
Vehicle(solvent)control	3h/20h	100	3.0
2000	3h/20h	19	NE
1500	3h/20h	14	NE
1000	3h/20h	8	NE
875	3h/20h	4	NE
750	3h/20h	7	NE
625	3h/20h	34	2,5
500	3h/20h	61	3,5
375	3h/20h	81	2,0
250	3h/20h	96	1,5
125	3h/20h	89	NE
62,5	3h/20h	92	NE
31,25	3h/20h	88	NE
Positive control	3h/20h	59	85,7***

Vehicle (solvent) control: 1% (v/v) DMSO

Positive control (+S9): Cyclophosphamide, 6μg/mL

NE: not evaluated

#: compared to the vehicle (solvent control)

##: in the final treatment medium at the end of the treatment

###: excluding gaps

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

It was concluded that the single result at 625 μg/mL with metabolic activation seen in a single replicate (not in the other replicate) was not reproducible in a repeat assay and did not show a dose response relationship. Hence this single result was not considered to represent an adverse effect of the test item.

The occurrence of polyploid and endoreduplicated metaphases was recorded in the main tests. Polyploid (1-11) and endoreduplicated (1-4) metaphases were found in some cases in the vehicle (solvent) control, positive control or test item treated samples in the performed experiments.

Applicant's summary and conclusion

Conclusions:

negative without and with metabolic activation (-/+ S9)